This application claims the benefit of priority under 35 U.S.C. § 119 to German application DE 103 15 640.2, filed Apr. 4, 2003.
In biomolecular processes it is often important to control the activity of an enzyme. This is particularly the case with DNA polymerase enzymes used for the polymerase chain reaction (PCR). PCR reactions oftentimes involve the use of a Mg2+-dependent heat-resistant DNA polymerase enzyme (such as Taq DNA polymerase) in a multi-cycle process employing several alternating heating and cooling steps to amplify DNA (U.S. Pat. Nos. 4,683,202 and 4,683,195). First, a reaction mixture is heated to a temperature sufficient to denature the double stranded target DNA into its two single strands. The temperature of the reaction mixture is then decreased to allow specific oligonucleotide primers to anneal to their respective complementary single stranded target DNAs. Following the annealing step, the temperature is raised to the temperature optimum of the DNA polymerase being used, which allows incorporation of complementary nucleotides at the 3′ ends of the annealed oligonucleotide primers thereby recreating double stranded target DNA. Using a heat-stable DNA polymerase, the cycle of denaturing, annealing and extension may be repeated as many times as necessary to generate a desired product, without the addition on polymerase after each heat denaturation. Twenty or thirty replication cycles can yield up to a million-fold amplification of the target DNA sequence (“Current Protocols in Molecular Biology,” F. M. Ausubel et al. (Eds.), John Wiley and Sons, Inc., 1998).
Although PCR technology has had a profound impact on biomedical research and genetic identity analysis, amplification of non-target oligonucleotides and mispriming on non-target background DNA, RNA, and/or the primers themselves, still presents a significant problem. This is especially true in diagnostic applications where PCR is carried out in a milieu of complex genetic backgrounds where the target DNA may be proportionately present at a very low level (Chou et al., Nucleic Acid Res., 20:1717-1723 (1992).
A chief problem is that even though the optimal temperature for Taq DNA polymerase activity is typically in the range of 62°-72° C., significant activity can also occur between 20°-37° C. (W. M. Barnes, et al, U.S. Pat. No. 6,403,341). As a result, during standard PCR preparation at ambient temperatures, primers may prime extensions at non-specific sequences because only a few base pairs at the 3′-end of a primer which are complementary to a DNA sequence can result in a stable priming complex. As a result, competitive or inhibitory products can be produced at the expense of the desired product. Thus, for example, structures consisting only of primers, sometimes called “primer dimers” can be formed by Taq DNA polymerase activity on primers inappropriately paired with each other.
The probability of undesirable primer-primer interactions also increases with the number of primer pairs in a reaction, particularly in the case of multiplex PCR. Mispriming of template DNA can also result in the production of inhibitory products or “wrong bands” of various lengths. During PCR cycling, non-specific amplification of undesired products can compete with amplification of the desired target DNA for necessary factors and extension constituents, such as dNTPs, which can lead to misinterpretation of the assay. Given the sensitivity of Taq DNA polymerase and its propensity to progressively amplify relatively large amounts of DNA from any primed event, it is imperative to control Taq DNA polymerase activity to prevent production of irrelevant, contaminating DNA amplification products, particularly when setting up PCR reactions.
Undesirable PCR side reactions typically occur during PCR preparation at ambient temperatures. One approach for minimizing these side reactions involves excluding at least one essential reagent (dNTPs, Mg2+, DNA polymerase or primers) from the reaction until all the reaction components are brought up to a high (e.g., DNA denaturation) temperature; the idea is to prevent binding of primers to one another or to undesired target sequences (Erlich, et al, Science 252, 1643-1651, 1991; D'Aquila, et al, Nucleic Acids Res. 19, 3749, 1991). This is an example of a “physical” PCR hot-start approach where an essential component is physically withheld until a desired reaction temperature is reached.
Other physical hot-start approaches have been described that physically segregate the reaction components from each other to guarantee that DNA polymerase activity is suppressed during the period preceding PCR initiation (see e.g., U.S. Pat. No. 5,643,764; Russian patent RU 2,215,037) or that employ the “chemical/biochemical hot-start” methods that utilize modified DNA polymerases reversibly activatable upon heating (e.g., AMPLITAQ GOLD™ DNA POLYMERASE, PE Applied Biosystems) or monoclonal, inactivating antibodies against Taq DNA polymerase that are bound to the polymerase at ambient temperatures (Scalice et al., J. Immun. Methods, 172: 147-163, 1994; Sharkey et al., Bio/Technology, 12:506-509, 1994; Kellogg et al., Biotechniques, 16: 1134-1137, 1994).
The different PCR hot-start approaches have multiple shortcomings. Physical hot-start methods are plagued by contamination problems, plugging up of pipet tips with wax or grease and increased heating times. Chemical/biochemical hot-start methods can damage the template DNA and can require prohibitively excessive amounts of expensive anti-Taq antibodies.
Accordingly, there is a need in the art for new PCR hot-start methods minimizing or eliminating the many problems or shortcomings associated with the prior art procedures. More generally, there is a need for new approaches for controlling other Mg-dependent enzymes or other non-Mg-dependent enzymes where controlled activity is desired.